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oxidative stress


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#1 dan_

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Posted 22 June 2009 - 09:12 AM

Hi everyone,

I would like to know if somebody has ever quantified oxidative stress in culture cells. If yes, where I can find a protocol/litterature?

Thanks!

#2 warsel

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Posted 23 June 2009 - 07:40 AM

Hi everyone,

I would like to know if somebody has ever quantified oxidative stress in culture cells. If yes, where I can find a protocol/litterature?

Thanks!


There are several ways, fluorimetric measurement of DCF-diacetate (fluorescent dichlorofluorescin) is what I used before.
It's a pretty straight forward assay, I don't have a protocol handy but I'm sure you'll find something online.

#3 Bill Nye

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Posted 23 June 2009 - 04:53 PM

There are several ways, fluorimetric measurement of DCF-diacetate (fluorescent dichlorofluorescin) is what I used before.
It's a pretty straight forward assay, I don't have a protocol handy but I'm sure you'll find something online.


I've used DCFDA before, try this on your cell line:

1) aspirate off media
2) incubate cells with 10uM dye in PBS @ 37c for 10mins
3) 2X wash with PBS
4) replenish media

- from my experience, DCF is leaky so try to work within 1hr after loading the dye
- my dye-stock is made in DMSO, I put some in PBS when I use the dye
- when loading the dye, use something without phenol red (ie PBS)

hope this helps :)

Edited by Bill Nye, 23 June 2009 - 04:53 PM.





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